Method of detecting cations using a tetra substituted cyclohexane

ABSTRACT

Disclosed is a tetra substituted cyclohexane having an ionophore in the one and two positions wherein the ionophore may be the same or different and is selected from the group consisting of a crown, a podand and a cryptand wherein the ionophore is capable of complexing with a metal or ammonium cation. Positions four and five are substituted by an electron donor group and an electron acceptor group. Also disclosed is a method for detecting the concentration of cations in a fluid by subjecting the fluid to the tetra substituted cyclohexane composition which will thereby complex the cation in the fluid. The cations are preferably alkali metal and alkaline earth metal cations. The fluid may be an aqueous or non-aqueous fluid or body fluids.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

The funding for work described herein was provided in part by theFederal Government, under a grant from the National Institute of Health.The government may have certain rights in this invention.

TECHNICAL FIELD

The present invention is concerned with the field of conformationallybiased intramolecular charge transfer complex containing ionophorecyclohexane compositions.

BACKGROUND OF THE INVENTION

Ionophores are materials capable of forming complexes with chargedspecies, preferably cations. Chromophores are chemical groups which whenpresent in a compound give color to the compound by causing adisplacement of, or appearance of, absorbance bands in the visiblespectrum.

The equilibrium constants for alkali metal complexation of polyethyleneglycol ethers can be measured by low temperature nuclear magneticresonance (NMR) spectroscopy by using a method which makes use ofconformational biasing. "Flipped-out" ionophores, such astrans-cyclohexano pentaethylene glycol diethyl ethers for thecomplexation of metal ions are disclosed in the Journal of The ChemicalSociety, Chemical Communications 1983, page 1409, M. Raban et al. Anoral presentation as to conformationally biased intramolecular chargedtransfer interaction was made in the Spring of 1989 at the Wayne StateUniversity, Department of Chemistry, Detroit, Mich. by D. Durocher et alwhich disclosed the ketal4-methylthiophenoxy-8,8-dimethyl-[4.3.0]-bicyclo-7,9-dioxo-nonane-3-ol-3,5-dinitrobenzoate.

The addition of barium and calcium to an azulene crown ether has beenreported as resulting in a shift in the UV-visible absorbance spectra ofthe material. Chem. Ber., 1984, 117, 2839, H. G. Lohr, F. Vogtle, H.Puff and W. Schuh. Since the donor atoms of the ionophore are notdirectly incorporated into the chromophore, the shift in spectra must bedue to the interaction of the cation with the negatively polarizedfive-membered ring in the azulene moiety. This interaction serves tostabilize the ground state and destabilize the excited state.

Chromoionophores have been developed that upon complexation exhibitbathochromic shifts, in the absorption maximum of the UV-visiblespectrum. For this to occur, the donor portion of the ionophore that isincorporated into the chromophore must be attached at its electron-poorend. Quinone imine ionophore shows a bathochromic shift upon theaddition of metal cations. Chem. Ber. 1981, 114, 638 J. Dix and F.Vogtle. An amino azulene chromoionophore shows significant bathochromicshift upon the addition of barium with the solution turning yellow toblue-violet. Chem. Ber. 1985, 118, 256, H. G. Lohr and F. Vogtle.Another chromoionophore that exhibits a large bathochromic shift in thepresence of pyridine/lithium is an azophenol crown reported inTetrahedron Lett., 1981, 22, 4407, T. Kaneda, K. Sugihara, H. Kamiya andS. Mitsumi. On addition of lithium chloride, the yellowish solutionturns deep purple. Other alkali metal cations show no change in thespectra. The material azophenol cryptand shows a high selectivity forpotassium in dioxane/morpholine as reported in European patentapplication 83100281. The color change allows the quantitativedetermination of potassium in solution or in test strips. In both cases,the material forms a neutral complex with the metal ion in the presenceof base.

It has been reported that the synthesis and characterization ofchromogenic spherand ionophores have high affinity for alkaline metalcations. Spherand chromophores exhibit a high affinity for bothpotassium and sodium in 80% dioxane / 20% water upon addition of1,5-diazabicyclo[4.3.0]non-5-ene, Journal of American Chemical Society,1988, 110, 571 D. J. Cram, R. A. Carmack and R. C. Helgeson. Cram hasalso reported the synthesis and characterization of mixed spherocryptand and sphero-crown type chromogenic ionophores. Journal ofAmerican Chemical Society, 1989, 111, 6339 R. C. Helgeson, B. P. Chech,E. Schapotau, C. R. Gebauer, A. Kumar and D. J. Cram.

None of the prior art demonstrates materials that have the capability ofacting as both a chromophore and an ionophore whereby the ionophore canpreferentially interact with metal ions and where there is a colorchange in the solution of the material but where the ionophore andchromophore are spacially separated and electronically insulated andwhere the color change derives primarily from a conformational changeattendant upon complexation.

SUMMARY OF THE INVENTION

Described are tetrasubstituted cyclohexanes having ionophoric group(s)in the 1,2 position and chromophores in the 4,5 position wherein theionophore groups are the same or different and may be a crown, podand orcryptand and are capable of complexing a metal ion or ammonium;

and wherein one chromophore is an electron donor moiety and the other isan electron acceptor moiety.

The invention is also concerned with the method of detecting cations ina fluid such as liquid comprising the steps of:

providing a cation or ammonium ion containing fluid such as liquid; and

complexing the ion from the fluid by treating the fluid with aneffective complexing amount of the above-identified tetra substitutedcyclohexane composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 are schematic drawings of the techniques for preparingcompounds of the present invention;

FIG. 3 is a chart of absorbance versus wavelength of products of theinvention; and

FIGS. 4 and 5 are charts of absorbance versus concentration of productsof this invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The tetra substituted cyclohexane compositions of the present inventionare designed to have an intramolecular charge transfer chromogen andionophore. Basically, the purpose of the composition is to complex thevarious metal ions such as alkali metal ions, alkaline earth metal ionsand ammonium ions and to effect a change in the UV-visible spectrum uponcomplexation. The alkali metal ions include lithium, sodium, potassiumand the like. The alkaline earth metal ions include calcium, magnesium,strontrum, barium and the like.

Ammonium ions are the fluid soluble ammonium ions that result fromdissolving primary and secondary amines. The amine containing materialsare alkyl amines, aralkyl amines and the hydroxy and carboxy derivativesthereof of from 6-20 carbon atoms. Suitable amines are para-3-aminopropyl benzyl amine; amino acids may be cystine, phenylalanine,tyrosine, halogenated tyrosines, thyronine, halogenated thyronines,proline, tryptophan and the like.

Synthetic ionophores are materials that are capable of forming complexeswith charged species, primarily cations. The common features shared byall ionophores are 1) the presence of an array of heteroatoms containingelectron pairs capable of forming donor-acceptor complexes with cationsand 2) a flexible hydrocarbon backbone capable of solvating the complexin both polar and nonpolar solvents. The geometry of the hydrocarbonbackbone contained in synthetic ionophores facilitates theirclassification into three categories: acyclic podands (i.e. polyethers),monocyclic coronands (i.e. crown ethers) and multicyclic cryptands.Examples of each class are: pentaethylene glycol diethyl ether,18-crown-6, an elipsoidal [2.2.2] cryptand, kryptofix-S,tetrathia-12-crown-4, and a spherical cryptand.

This case is further concerned with charge transfer complexes orswitchable intramolecular charge-transfer complexes (sometimes calledelectron donor-acceptor complexes, Mulliken, R. S., J. Am. Chem. Soc.1952, 74, 811, herein incorporated by reference). These complexes giverise to absorption bands that are not present in the two parentcompounds that form the complex. The intensity of these absorption bands(and the extent of intramolecular complexation) can be controlled bychanging the relative molecular geometry of the complex framework, whichis accomplished by chemical manipulation of the parent compounds.

The interaction of a molecule of high electron affinity with anothermolecule possessing low ionization potential may give rise to amolecular complex (or charge transfer complex) possessing properties notfound in either component substance. These properties are: 1) newelectronic absorption bands, 2) a loss in diamagnetic susceptibility,and 3) an increase in paramagnetic susceptibility (the presence ofunpaired electrons).

The purpose of the compositions is to permit the detection of these ionsin fluids such as aqueous or non-aqueous liquids, mammallian bodyliquids such as blood, urine and the like and other liquid fluids inwhich the metal ions are dissolved such as liquid hydrocarbons, liquidhalogenated hydrocarbons from one to six carbon atoms, aliphaticalcohols or esters, aliphatic glycols or glycol ethers, heterocyclicsolvents such as dioxane, furan, pyridine and mixtures thereof.

Having described the invention, listed below are preferred embodimentswherein all parts are parts by weight and all temperatures are indegrees Centigrade unless otherwise indicated.

EXAMPLE 1 Synthesis of 1(S,R), 2(S,R), 4(S,R),5(S,R)-2-(4-methylthiophenoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dinitrobenzoate

The product was a result of the stepwise reaction scheme illustrated inFIG. 1.

The reaction steps proceeded as follows.

A. 1,4-Cyclohexadiene (50.0 g, 0.626 mol) was added to a mixture of 225ml of distilled water and 25 ml of ρ-dioxane. The mixture was stirred at25° C. and N-bromo succinimide (108.9 g, 0.612 mol) was added slowlyover a 1 hour period. The mixture was allowed to stir an additional 3hours. The reaction mixture was then extracted 3 times with 100 mlportions of chloroform. The combined organic layers were then extracted3 times with 50 ml portions of saturated aqueous sodium bicarbonate thendried over sodium sulfate and filtered. The solvent was removed in vacuoand the crude bromohydrin was immediately added to 1050 ml of a freshlyprepared 1.0 N sodium hydroxide solution and allowed to stir for 1 hourat 25° C. The reaction mixture was extracted three times with 100 mlportions of chloroform and the combined organic layers were dried oversodium sulfate and filtered. The solvent was removed in vacuo giving thecrude epoxide which was distilled at 5 mm Hg. The fraction collected at38°-40° C. (58.5 g, 97%) was shown to be subreaction product 1: [4.1.0]Bicyclo-7-oxo-hept-3-ene.

B. To 126 ml of a 6N solution of potassium hydroxide was added 15 ml ofρ-dioxane and 123.4 g (243.6 mmol) of [4.1.0] Bicyclo-7-oxo-hept-3-ene.The stirred mixture was allowed to reflux for 24 hours and then cooledto room temperature. 150 ml of brine was added and the mixture wasextracted 7 times with 50 ml aliquots of chloroform. The combinedorganic layers were dried over sodium sulfate and the solvent wasremoved in vacuo. The resulting oil solidified upon standing giving thecrude diol. The diol was dissolved in hot ethyl acetate, decolorizedwith activated charcoal and recrystallized from hot 75% ethylacetate/25% heptane to give subreaction product 2,[1(R,S),2(R,S)]4-cyclohexen-1,2-diol as white crystals, 15.9 g (57.4%).

C. A 3 neck flask was fitted with a magnetic stirrer, a condenser and anaddition funnel. The flask was flame dried and constantly purged withdry nitrogen. To the flask, 300 ml of dry THF and sodium hydride (2.78g, 1.1 equiv., 50% oil dispersion, washed twice with hexane) were addedand allowed to stir. [1(R,S),2(R,S)]4-Cyclohexen-1,2-diol (3.0 g, 26.3mmol) and 18-crown-6 (0.10 g, catalyst) were added to the solution andstirred for 4 hours. The flask was put in a brine-ice bath (-5° C.) and2-(2-ethoxyethoxy)ethyl ρ-toluene sulfonate (17.42 g, 1.15 equiv.) wasdissolved in 50 ml of dry THF and added dropwise over a 2 hour period.The solution was warmed to room temperature and the flask was fittedwith a heating mantle. The solution was heated and allowed to reflux for48 hours, then cooled. The mixture was filtered and the solvent removedin vacuo. The product was separated by flash column chromatography (300g silica gel, 40% ethyl acetate/60% petroleum ether rising to 75% ethylacetate/25% petroleum ether, TLC:R_(f) diol=0.31, R_(f) product =0.39,in 50/50 EthOAc/Pet Eth) giving subreaction product 3,4(S,R),5(S,R)-4,5-bis(1,4,7-trioxononanyl)-cyclohex-1-ene as aclear-yellowish oil (3.7 g, 41%).

The 2-(2-ethoxyethoxy)ethyl ρ-toluene sulfonate was prepared as follows:Pyridine (75 ml) was added to a flask containing ρ-toluene sulfonylchloride (16.0 g, 0.085 mol) and swirled until the solid dissolved. Themixture was cooled to 0° C. and 2-(2-ethoxyethoxy) ethanol (11.4 g,0.085 mol) was added. The mixture was stirred for 3 hours and 10%sulfuric acid added until the pH of the solution was 5 or less. Thesolution was then extracted 3 times with ether and the combined organiclayers were dried over sodium sulfate. The solvent was removed in vacuoleaving an oil that was shown to be pure 2-(2-ethoxyethoxy)ethylρ-toluene sulfonate (16.1 g, 55%, 0.047 mol).

D. A 250 ml flask was flame dried and fitted with a drying tube and amagnetic stirrer. The flask was then charged with 125 ml of methylenechloride, 4(S,R),5(S,R)-4,5-bis(1,4,7-trioxononanyl)-cyclohex-1-ene-(1.5g, 4.33 mmol) and chilled in an ice bath. The solution was allowed tostir for 15 minutes, then meta-chloroperoxy-benzoic acid (80%, 1.51 g,8.5 mmol) was added and the solution was stirred for 2 hours. Thereaction mixture was then warmed slowly to room temperature and allowedto stir overnight. The solvent was removed in vacuo while maintainingthe bath temperature below 30° C. and the remaining oil was dissolved in100 ml of ether. The organic solution was extracted 3 times with 20%aqueous sodium bisulfite, 3 times with saturated sodium bicarbonate andonce with brine. The organic layer was dried over sodium sulfate and thesolvent removed in vacuo. The crude oil was purified using flashchromatography (150 g silica gel, 40% ethyl acetate/60% petroleum etheryielding 1.263 g (64%) 1(R,S),2 (S, R),4 (S,R),5(S,R)-4,5-bis(1,4,7-trioxononanyl)-cyclohex-1-ene oxide (subreaction product 4), as aclear, white oil.

E. A 100 ml flask was flame dried and purged with dry nitrogen. Theflask was fitted with a condenser, magnetic stirrer and charged with 25ml of tert-butyl alcohol. 4-methylthiophenol (4 equivalents) wasdissolved in 25 ml of dry THF and added to the reaction flask. To thesolution, potassium tert-butoxide (0.747 g, 4.0 equivalents) was addedand the solution was allowed to stir for 20 minutes.1(R,S),2(S,R),4(S,R),5(S,R)-4,5-bis (1,4,7-trioxononanyl)-cyclohex-1-eneoxide (0.655 g, 1.81 mmol) was added to the mixture, the flask wasfitted with a heating mantle and the mixture was warmed and allowed toreflux for 4 days. The solvent was removed in vacuo and the residue wasdissolved in diethyl ether. The organic solution was extracted 3 timeswith 10% sodium hydroxide, once with brine and dried over magnesiumsulfate. The solvent was removed in vacuo leaving pure2-(4-methylthiophenoxy)-4,5-bis(1,4,7-trioxanonanyl)-1cyclohexanol(subreaction product 5) as a clear oil (92-97%).

F. A 50 ml flask was flame dried and fitted with a magnetic stirrer anda drying tube. Dry benzene (25 ml), pyridine (0.554 g, 3 equivalents),4-dimethylaminopyridine (DMAP, 0.10 g, catalyst) and2-(4-methylthiophenoxy-4,5-bis (1,4,7-trioxanonanyl)-1-cyclohexanol(1.755 mmol) were added and allowed to stir for 5 minutes until all ofthe added DMAP had dissolved. To the solution, 3,5-dinitrobenzoylchloride (1.22 g, 3.9 equivalents) was added and the mixture was stirredfor 24 hours. The solution was filtered through Celite and the solventwas stripped in vacuo. The crude oil was purified by columnchromatography with 50/50 ethyl acetate/pet ether escalating to 100%ethyl acetate after 1 column volume. Final product 6,2-(4-methythiophenoxy)-4,5-bis (1,4,7-trioxanonanyl) cyclohexyl-3,5-dintrobenzoate, was isolated as an orange oil in moderate yields(32-54%).

EXAMPLE 2 Synthesis of 1(S,R), 2(S,R), 4(S,R),5(S,R)-2-(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dinitrobenzoate

The reaction synthesis proceeded as in Example 1 until subreaction E.Four equivalents of 2-napthol were added in place of the4-methylthiophenol. After subreaction E produced the correspondingalcohol (subproduct 5), 2-(2-napthoxy)-4,5-bis(1,4,7trioxanonanyl)-1-cyclohexanol, subreaction F proceeded as in Example 1and produced2-(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dintrobenzoate,final product 7, which was purified and isolated as an orange or yellowoil in moderate yield (32-54%).

EXAMPLE 3 Synthesis of 1(S,R), 2(S,R), 4(S,R),5(S,R)-2-(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3-nitrobenzyl ether

The reaction proceeded as in Example 2 for the synthesis of2-(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-1-cyclohexanol.

A 100 ml flask was then flame dried, purged with dry nitrogen and fittedwith a magnetic stirrer, condenser and dropping funnel. Dry THF (25 ml)was added to the flask and subproduct 5, 2-(2-napthoxy)-4,5bis(1,4,7-trioxanonanyl)-1-cyclohexanol (0.400 g, 0.797 mmol), and sodiumhydride (0.0765 g, 2.0 equiv., 50%, unwashed) were added and the mixturewas stirred for 4 hours. The reaction flask was immersed in a brine icebath and cooled to -5° C. To the chilled solution, 3-nitrobenzyl bromide(0.362 g, 2.1 equiv.) dissolved in 25 ml of dry THF, was added over a 4hour period. The reaction mixture was stirred at 0° C. for an additional4 hours, then allowed to warm to room temperature. The mixture was thenwarmed and allowed to reflux for 24 hours. The solution was cooled,filtered through a Celite pad and the solvent removed in vacuo. Thecrude product was purified by column chromatography using 50/50 ethylacetate/pet ether (R_(f) (prod)=0.41 on TLC) yielding pure2-(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3-nitrobenzylether, final product 8, as a yellow oil.

EXAMPLE 4 Synthesis of 1(R,S), 2(R,S), 4(R,S),5(R,S)-4,5-bis-((dipropylcarbamoyl)-methoxy)-2-(4-methylthiophenoxy)-cyclohexyl-3,5-dinitrobenzoate

The product was a result of the stepwise reaction scheme illustrated inFIG. 2. The reaction steps proceeded as follows.

A. 1,4-Cyclohexadiene (50.0 g, 0.626 mol) was added to a mixture of 225ml of distilled water and 25 ml of ρ-dioxane. The mixture was stirred at25° C. and N-bromo succinimide (108.9 g, 0.612 mol) was added slowlyover a 1 hour period. The mixture was allowed to stir an additional 3hours. The reaction mixture was then extracted 3 times with 100 mlportions of chloroform. The combined organic layers were then extracted3 times with 50 ml portions of saturated aqueous sodium bicarbonate thendried over sodium sulfate and filtered. The solvent was removed in vacuoand the crude bromohydrin was immediately added to 1050 ml of a freshlyprepared 1.0N sodium hydroxide solution and allowed to stir for 1 hourat 25° C. The reaction mixture was extracted three times with 100 mlportions of chloroform and the combined organic layers were dried oversodium sulfate and filtered. The solvent was removed in vacuo giving thecrude epoxide which was distilled at 5 mm Hg. The fraction collected at38°-40° C. (58.5 g, 97%) was shown to be [4.1.0]Bicyclo-7-oxo-hept-3-ene, subreaction product 9.

B. To 126 ml of a 6N solution of potassium hydroxide was added 15 ml ofρ-dioxane and 123.4 g (243.6 mmol) of [4.1.0] Bicyclo-7-oxo-hept-3-ene.The stirred mixture was allowed to reflux for 24 hours and then cooledto room temperature. 150 ml of brine was added and the mixture wasextracted 7 times with 50 ml aliquots of chloroform. The combinedorganic layers were dried over sodium sulfate and the solvent wasremoved in vacuo. The resulting oil solidified upon standing giving thecrude diol, which was dissolved in hot ethyl acetate, decolorized withactivated charcoal and recrystallized from hot 75% ethyl acetate/25%heptane to give subreaction product 10; [1(R,S),2(R,S)] 4Cyclohexen-1,2-diol, as white crystals, 15.9 g (57.4%): mp 97°-9° C.

C. A 500 ml flask was flame dried and fitted with a mechanical stirrer,condenser, and addition funnel. The flask was charged with 125 ml dryTHF and [1(R,S),2(R,S)] 4 Cyclohexen-1,2-diol (4.0 g, 35 mmol) was addedand stirred until all of the added solid had dissolved. To the solution,18-crown-6 (0.200 g) and sodium hydride (6.90 g, 2.1 equivalents, 50%washed twice with hexane) were added and the mixture was stirred for 4hours at room temperature. Bromoacetic acid (9.75 g, 2.0 equivalents,85%) was dissolved in 100 ml of dry THF, transferred to the additionfunnel and added dropwise to the reaction mixture over a 4 hour period.The dropping funnel was removed and the reaction flask was fitted with aheating mantle. The reaction mixture was warmed and allowed to refluxfor 48 hours. The solvent was removed in vacuo and the residue wasdissolved in 50 ml of saturated sodium bicarbonate. Concentratedhydrochloric acid was added to the solution until acidic to litmus(CAUTION: gas evolution) and the acidic aqueous solution was extracted 3times with ether. The combined organic extracts were dried overmagnesium sulfate and the solvent was removed in vacuo. The crude diacidwas recrystallized from hot ethyl acetate yielding pure1,(R,S),2(R,S)-1,2-bis(carboxymethoxy)-4-cyclohexane, subreactionproduct 11, as white crystals (27%). m.p. 87.5°-90° C.

D. A 50 ml flask was flame dried and fitted with a magnetic stirrer anddrying tube. Dry benzene (10 ml), dry THF (10 ml) and1,(R,S),2(R,S)-1,2-bis(carboxymethoxy)-4-cyclohexane (0.900 g, 3.91mmol) were added to the flask and stirred until all of the added solidhad dissolved. Thionyl chloride (1.384 g, 3.0 equiv.) was added and themixture was stirred for 20 hours. The solvent was removed in vacuo anddry benzene (25 ml) was added. The process was repeated two more times,then a final addition of dry benzene (20 ml) was added and the reactionmixture was chilled to 0° C. in an ice bath. Dipropyl amine (2.764 g,7.0 equiv.) was added and the solution was stirred for an additional 24hours. The solvent was removed and the residue was dissolved in 50 ml ofchloroform and extracted three times with 1N HCl, three times withsaturated aqueous sodium bicarbonate and the layers separated. Theorganic layer was dried over magnesium sulfate, filtered and the solventremoved in vacuo leaving 1(R,S),2(R,S)-1,2-bis (dipropylcarbamoyl)methoxy-4-cyclohexene, subreaction product 12, (0.974 g, 63%) as ayellow oil.

E. A dry 100 ml flask was charged with methylene chloride (50 ml) andchilled in an ice bath to 0° C. 1(R,S),2(R,S)-1,2-bis(dipropylcarbamoyl)methoxy-4-cyclohexene (0.974 g, 2.53 mmol) and meta-chloroperoxy-benzoicacid (1.31 g, 3.0 equiv., 85% (tech)) were charged to the flask andstirred for 8 hours at 0° C. The reaction mixture was allowed to standat room temperature overnight then the solvent was removed in vacuo withthe bath temperature not exceeding 30° C. The residue was dissolved indiethyl ether (50 ml) and the solution was extracted three times with20% aqueous sodium bisulfite, three times with saturated sodiumbicarbonate and once with brine. The organic layer was dried over sodiumsulfate, filtered and the solvent removed in vacuo. The epoxide waspurified by column chromatography using 90/10 ethyl acetate/petroleumether yielding the product of subreaction E as pure1(R,S),2-(S,R),4(R,S),5(R,S)-4,5bis((dipropylcarbamoyl)-methoxy-4-oxa-[4.1.0]-bicycloheptane(subreaction product 13) as a clear oil (0.48 g, 45%.

F. A 100 ml flask was flame dried, purged with dry nitrogen and fittedwith a magnetic stirrer, heating mantle and condenser. Dry THF (25 ml),t-butanol (25 ml), potassium t-butoxide (0.700 g, 2.5 equiv.) and4-methylthiophenol (1.0515 g, 3.0 equiv.) were added and the mixture wasallowed to stir until all of the added solid had dissolved.1,(R,S),2,(S,R),-4(R,S),4(R,S),5(R,S)-4,5-bis((dipropylcarbamoylmethoxy)-4-oxa-[4.1.0]-bicycloheptane (1.030 g, 2.50mmol) was added and the mixture was warmed and allowed to reflux for 72hours. The reaction vessel was cooled to room temperature and thesolvent was removed in vacuo. The residue was dissolved in diethyl etherand extracted 4 times with saturated sodium carbonate and once withbrine. The organic layer was dried over sodium sulfate, filtered and thesolvent was removed in vacuo. Pure product F was obtained by columnchromatography of the residue with 100% ethyl acetate yielding pure1,(R,S),2(R,S),4(R,S),5(R,S)-4,5-bis(dipropylcarbamoyl)-methoxy)-2-(4-methyl thiophenoxy)-cyclohexanol,subreaction product 14, (0.423 g, 31%) as a viscous oil.

G. A 50 ml flask was flame dried, fitted with a magnetic stirrer and adrying tube and charged with dry benzene (25 ml). Pyridine (0.242 g, 4.0equiv.), dimethylaminopyridine (DMAP, 0.0050 g, cat) and1,(R,S),2(R,S),4(R,S),5(R,S)-4,5-bis(dipropylcarbamoyl)-methoxy)-2-(4-methylthiophenoxy)-cyclohexanol (0.423 g, 0.7663 mmol) were charged to theflask and allowed to stir until all of the added materials haddissolved. To the mixture, 3,5-dinitrobenzoyl chloride (0.530 g, 4.0equiv.) was added and the solution was stirred for 72 hours. Thesolution was filtered and then extracted three times with 1N (HCl, threetimes with saturated sodium bicarbonate and once with brine. Thesolution was dried over sodium sulfate, filtered and the solvent removedin vacuo leaving final product 15,1(R,S),2(R,S),-4R,S),5(R,S),4,5-bis((dipropylcarbamoyl)-methoxy)-2-(4-methylthiophenoxy-cyclohexyhexyl-3,5-dinitrobenozateas a yellow-orange oil which was further purified by columnchromatography with ethyl acetate (0.545 g, 95%).

EXAMPLE 5 Complexation of Na⁺¹, K⁺¹, Rb⁺¹ and Cs⁺¹ by: 1(S,R), 2(S,R),4(S,R, 5(S,R)-2(4-methylthiophenoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dinitrobenzoate, 1(S,R), 2(S,R),4(S,R),5(S,R)-2(2-napthoxy)-4,5-bis-(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dinitrobenzoate,and 1(S,R), 4(S,R), 5(S,R)-2(2-napthoxy)-4,5-bis(1,4,7-trioxanoanyl)-cyclohexyl-3nitrobenzyl ether

A known amount of a charge transfer ionophore was dissolved in a solventsuch as methanol and a premeasured amount of solid salt was added to thesolution. Suitable salts include sodium nitrate, potassium bromate,potassium chloride and bromide, rubidium perchlorate and cesiumchloride. The vial was shaken vigorously for about two minutes and theUV-visible absorption spectrum was taken. This was then subtracted froma spectrum of the free ionophore.

The effect of the addition of 1.0 equivalent of salt to 0.0050Msolutions of the three ionophores: 1(S,R), 2(S,R), 4(S,R),5(S,R)-2(4-methylthiophenoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3,5-dinitro-benzoate, 1(S,R), 2(S,R),4(S,R), 5(S,R)-2(2-napthoxy)-4,5-bis(1,4,7trioxanonanyl)-cyclohexyl-3,5-dinitrobenzoate, and 1(S,R), 2(S,R),4(S,R), 5(S,R)-2(2-napthoxy)-4,5-bis(1,4,7-trioxanonanyl)-cyclohexyl-3nitrobenzyl ether, is plotted as FIG.5.

EXAMPLE 6

Complexation of Ca⁺², Mg⁺² and Sr⁺² by: 1(S,R), 2(S,R), 5(S,R)-4,5-bis((4-dipropylcarbamoyl)-methoxy)-b2-(4-methylthiophenoxy)-cyclohexyl-3,5-dintrobenzoate

A known amount of 1(S,R), 2(S,R), 5(S,R)-4,5-bis((4-dipropylcarbamoyl)-methoxy)-2-(4-methylthiophenoxy)-cyclohexyl-3,5-dintrobenzoatewas dissolved in a solvent such as methanol, acetonitrile, ornitromethane. A premeasured amount of solid salt was added to thesolution. Suitable salts include magnesium nitrate hexahydrate, calciumnitrate tetrahydrate and strontium nitrate. The vial was shakenvigorously for about two minutes and the UV-visible absorption spectrumwas taken. This was then subtracted from a spectrum of the freeionophore.

The effect of salt addition to a 0.0050M solution of 1(S,R), 2(S,R),5(S,R)-4,5-bis((4-dipropylcarbamoyl)-methoxy)-2-(4-methylthiophenoxy)-cyclohexyl-3,5-dintrobenzoate methanolis summarized on FIG. 3 and 4.

As can be appreciated from FIGS. 1 and 2 and the above examples, thepreparation of desired electron acceptor and/or donor groups can beinserted as various reactants to make them present in the final product.The electron donor and acceptor reactants are preferably from 6 to 20carbon atoms. Examples of suitable electron acceptor/donors are listedbelow with reference to a desirable reactant in FIGS. 1-2:

Electron Acceptors

Acyl

4-acetylbenzoyl chloride

3-acylbenzoyl chloride

Cyano

3-cyano-benzyl chloride

3-cyano-benzyl bromide

Nitro

4-nitrobenzyl chloride

Nitrophenyl halides

2-4-dinitro-1-chloro-benzene

    ______________________________________                                        Desired Reactants     Figure #,                                               For Above Electron Acceptors                                                                        Product #                                               ______________________________________                                        2-(4-methylethiophenoxy-                                                                            1, 5                                                    4,5-bis-(1,4,7-trioxanonanyl)-                                                2-cyclohexanol                                                                2-(2-napthoxy)-4,5-bis-(1,4,7-                                                                      1, 5                                                    trioxanonanyl)-1-cyclohexanol                                                 4,5-bis(dipropylcarbamoyl)-                                                                          2, 14                                                  methoxy)-2-(4-methylthiophenoxy)-                                             cyclohexanol                                                                  ______________________________________                                    

Electron Donors

Amino Acid

Tryptophan (11 carbons)

Substituted Amines

3-(n,n-dimethyl)-aminophenol

Amidic Groups

4-acetamidophenol

Alkyl Thio

4-methylthiophenol

Hydroquinone

resorcinol

Mercaptophenol

3-mercaptophenol

    ______________________________________                                        Desired Reactants    Figure #,                                                For Above Electron Donors                                                                          Product #                                                ______________________________________                                        1(R,S), 2(S,R), 4(S,R)                                                                             1, 4                                                     5(S,R)-4,5-bis(1,4,7-                                                         trioxonanyl)-cyclohex-1-ene                                                   oxide                                                                         1(R,S), 2(S,R), 4(R,S),                                                                             2, 13                                                   5(R,S)-4,5-bis(dipropylcaramoyl)-                                             methoxy)-4-oxa[4.1:0]                                                         bicycloheptane                                                                ______________________________________                                    

It is contemplated that the invention can be utilized in the followingmanner to quantitatively determine the concentration of Ca⁺² in a sampleof human or mammalian body fluid.

An organic extraction, using a suitable, non-H₂ O miscible organicsolvent such as chloroform, methylene chloride, or ether containing atetra substituted cyclohexane composition, will be performed on a sampleof body fluid. After separation, the organic mixture will be subjectedto a UV-spectrophotometer. Known amounts of Ca⁺² will be added to samplealiquots of the organic mixture. A plot of absorbance versus [Ca⁺² ]will allow the determination of the [Ca⁺² ] of the body fluid viainterpolation or extrapolation.

While the forms of the invention herein disclosed constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is understood that the terms used herein are merelydescriptive rather than limiting, and that various changes may be madewithout departing from the spirit or scope of the invention.

What is claimed is:
 1. A method of detecting cations in a liquidcomprising the steps of:a) providing a liquid containing metal orammonium ions; b) complexing the ion from the liquid by treating theliquid with an effective complexing amount of a tetra substitutedcyclohexane composition; wherein the tetra substituted cyclohexane hasan ionophore in the 1,2 positions wherein the ionophore may be the sameor different and is selected from the group consisting of a crown, apodand and a cryptand; and wherein positions 4,5 are substituted by anelectron donor group and an electron acceptor group.
 2. The method ofclaim 1 wherein the cyclohexane is in the chair form.
 3. The method ofclaim 2 wherein the ionophore of the chair form is the diequatorialisomer.
 4. The method of claim 2 wherein the ionophore is the diaxialposition.
 5. The method of claim 2 wherein the tetra substitutedcyclohexane has the following structure: ##STR1## wherein A is theionophore and B and C is the electron donor or the electron acceptorgroups.
 6. The method of claim 2 wherein the electron acceptor group isa nitro substituted benzoyl.
 7. The method of claim 2 wherein theelectron donor is an araloxy group of from six to twenty carbon atoms.8. The method of claim 2 wherein the electron donor is a thioaryl groupof from six to twenty carbon atoms.
 9. The method of claim 2 wherein thetetra substituted cyclohexane has the following structure: ##STR2## 10.The method of claim 2 wherein the tetra substituted cyclohexane has thefollowing structure: ##STR3##
 11. The method of claim 2 wherein theliquid is a liquid from a mammal.
 12. The method of claim 2 wherein thecation is an alkali metal ion.
 13. The method of claim 2 wherein thecation is an alkaline earth metal cation.
 14. The method of claim 11wherein the ionophore is a crown.
 15. The method of claim 1 wherein theionophore is a podand.
 16. The method of claim 1 wherein the ionophoreis a cryptand.